SOMATIC EMBRYOGENESIS FROM LEAF CALLUS DERIVED FROM MATURE TREES OF THE CYCAD CERATOZAMIA-HILDAE (GYMNOSPERMAE)

A friable and transient embryogenic callus was initiated from pinnae removed from leaves in new vegetative flushes of mature Ceratozamia hildae Landry and Wilson, a cycad. Somatic proembryos developed from the callus approximately 3 months after explanting onto plant growth medium consisting of a modified B5 formulation with 60 g l(-1) sucrose, 400 mg l(-1) glutamine, 100 mg l(-1) arginine, 100 mg l(-1) asparagine, 4.5 mu M 2,4-dichlorophenoxyacetic acid with either 1.2 mu M or 4.6 mu M kinetin and 1.75 g l(-1) gellan gum. Following subculture of somatic proembryos at this time onto medium without plant growth regulators, they continued to proliferate by a process resembling cleavage embryony or polyembryogenesis for several months. Proliferating embryogenic cultures consisted of hyperhydric somatic proembryos. Some 15 months after explanting, the somatic proembryos began to change in appearance; the suspensors became white and opaque, but were usually highly branched due to cleavage embryony. A single cotyledonary somatic embryo usually developed from the tip of each of the suspensors. Somatic embryos were primarily dicotyledonous, and less frequently monocotyledonous. Fewer than 10% of the somatic embryos appeared to be morphologically abnormal. Germination occurred in vitro whereby the coleorhiza elongated and a tap root emerged; however, plantlet recovery has not been demonstrated because the shoot axis failed to elongate.